Generation of Replication-Dependent Double-Strand Breaks by the Novel N2-G-Alkylator S23906-1 Ste ´phane Le ´once, 1 Laurence Kraus-Berthier, 1 Roy M. Golsteyn, 1 Marie-He ´le `ne David-Cordonnier, 2 Christelle Tardy, 2 Ame ´lie Lansiaux, 2 Virginie Poindessous, 3 Annette K. Larsen, 3 and Alain Pierre ´ 1 1 Institut de Recherches Servier, Cancer Drug Discovery, Croissy sur Seine, France; 2 Institut National de la Sante´ et de la Recherche Me ´dicale (INSERM) U-524 and Centre Oscar Lambret, Institut de Recherche sur le Cancer de Lille, Lille, France; and 3 INSERM U-673 and Universite ´ Pierre et Marie Curie Paris 6, Ho ˆpital Saint-Antoine, Paris, France Abstract S23906-1, a new DNA alkylating agent that reacts with the exocyclic 2-NH 2 group of guanine residues yielding monofunc- tional adducts, is currently under clinical evaluation in phase I trials. To investigate the mechanism of action of S23906-1, we compared parental KB-3-1 cells and KB/S23-500 cells that are 15-fold resistant to S23906-1. Cell death induced by 1 Mmol/L S23906-1 in KB-3-1 cells was associated with their irreversible arrest in the G 2 -M phases of the cell cycle followed by apoptosis, whereas a proportion of the resistant KB/S23- 500 cells were able to exit from the G 2 arrest and divide, leading to a significantly lower rate of apoptosis. The attenuated apoptotic response was associated with decreased Chk2 protein phosphorylation, indicating that the DNA damage signaling pathways are more potently activated in the sensitive cells. However, similar rates of adduct formation and repair were measured in both cell lines. Exposure to S23906-1 induced a higher formation of DNA breaks, measured by the comet assay, in sensitive cells. In agreement, a histone H2AX phosphorylation assay revealed that S23906-1 induced double-strand breaks (DSB) in a dose- and time- dependent manner and that these were more persistent in the parental cells. These DSBs were found mainly in S-phase cells and inhibited by aphidicolin, suggesting that they are DNA replication-mediated DSBs. These results suggest that second- ary DNA lesions play an important role in the cytotoxicity of this compound and make histone H2AX phosphorylation an attractive marker for monitoring the efficacy of S23906-1. (Cancer Res 2006; 66(14): 7203-10) Introduction Acronycine, an alkaloid that was first isolated from the Australian shrub Sarcomelicope simplicifolia , was shown to exhibit a broad spectrum of antitumor activity in experimental models (1). However, the phase I/II clinical trials of acronycine were not conclusive due to both a low response rate as well as neurologic and gastrointestinal toxicities (2). The unstable acronycine epoxide, which was later isolated from Sarcomelicope argyrophylla , was assumed to be the active metabolite of the parent acronycine molecule (3). More stable prodrugs of this electrophilic epoxide, obtained by grafting two esters at the C1-C2 position of the pyran ring, were synthesized (4), leading to a family of diesters of benzo[b ]acronycine, which were markedly more potent than acronycine (5). From this series, the diacetate S23906-1 was identified as one of the most potent derivatives both in vitro (6) and in vivo in orthotopic models of human solid tumors (5, 7). S23906-1 is currently in phase I clinical trials. DNA was identified as an important target for this alkylating agent. We have previously shown, using DNA relaxation experi- ments done with supercoiled DNA, that S23906-1 neither affects the topoisomerase I–mediated relaxation of plasmid DNA nor intercalates into DNA, as shown by circular and linear dichroism measurements, but specifically reacts with the exocyclic 2-NH 2 group of guanine residues exposed in the minor groove of DNA to form a covalent adduct (8, 9). This compound is one of very few antitumor agents that induce monofunctional DNA adducts at the N 2 position of guanine: ecteinascidin 743, a marine product with a very different structure, being the most recent one (10). However, ecteinascidin 743 potently stabilizes duplex DNA against heat denaturation, whereas S23906-1 markedly destabilizes the duplex (11). To date, all the structure-activity relationships support DNA alkylation as the main molecular mechanism of action in the benzo[b ]acronycine series (12). However, the reactivity of S23906-1 to glutathione (13) suggests that additional nucleophilic targets other than DNA might be involved in its mechanism of action. At the cellular level, S23906-1 also presents uncommon properties, such as a selective increase in cyclin E, and a dual effect on cell cycle depending on the concentration (14). The aim of the present work was to investigate the cellular responses to S23906-1 to delineate some determinants for sensi- tivity to S23906-1. For this purpose, we used a sensitive parental cell line, KB-3-1, and a variant cell line, KB/S23-500, which was made resistant by stepwise exposure of KB-3-1 cells to S23906-1. Due to the fact that a similar number of primary DNA adducts were found in sensitive and resistant cells, we searched for secondary DNA lesions and investigated the relationship with induction of apop- tosis. We provide evidence, using the comet and histone H2AX phosphorylation assays, supporting the conversion of the primary adducts into DNA double-strand breaks (DSB). Materials and Methods Compounds. Acronycine and S23906-1 (Fig. 1) were synthesized as described (5). Both compounds were solubilized at 10 À2 mol/L in DMSO, aliquoted, and stored at À20jC. Cytarabine was provided by Pharmacia & Upjohn SA (St-Quentin-en-Yvelines, France); vincristine and gemcitabine by Eli Lilly (Saint-Cloud, France); cisplatin by Bellon-Aventis (Montrouge, Requests for reprints: Ste´phaneLe ´once, Institut de Recherches Servier, Division de Recherches Cancerologie, 125 Chemin de Ronde, 78290 Croissy sur Seine, France. Phone: 33-1-55-72-22-84; E-mail: stephane.leonce@fr.netgrs.com. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3946 www.aacrjournals.org 7203 Cancer Res 2006; 66: (14). 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